Mediucm for detecting Van A and Van B vancomycin-resistant entercocci and method of using the same

ABSTRACT

Van A and Van B vancomycin resistant  enterococci  detection media as well as a method of selectively detecting Van A and Van B vancomycin resistant  enterococci  clinically important in vancomycin resistant  enterococci  from testing microorganisms or specimens using the media. The media for selectively detecting Van A and Van B VRE from testing microorganisms and specimens are media where  enterococci  can grow where vancomycin, D-cycloserine and D-lactate are added. Preferably 32-256 μg/ml of vancomycin, 1-64 μg/ml of D-cycloserine, and 0.025-0.8 mol/l of sodium lactate are added to culture medium where  enterococci  can grow.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a medium for detecting Van A and Van Bvancomycin resistant enterococci and a method of detecting Van A and VanB vancomycin resistant enterococci by using the medium. Particularly,the invention relates to a medium with certain formula for detecting VanA and Van B vancomycin resistant enterococci and to a method ofselectively detecting clinically important Van A and Van B vancomycinresistant enterococci in test microorganisms or specimens by using saidmedium.

PRIOR ART

Enterococci, as its name implies, are catalase negative and Grampositive cocci, forming indigenous microbial flora in the intestine andincluding about 20 organisms. Conventionally, enterococci had beenclassified as a part of Streptococcus species but have been classifiedas an independent species, enterococci, because of DNA homology and 16Sribosomal RNA sequence analysis, etc. Clinically, enterococci areprimarily found in the urinary tract and the biliary tract, and areoften causative agents for sepsis and infectious endocarditis.Enterococcus faecalis is the most frequently isolated organism, followedby Enterococcus faecium and Entorococcus avium. Other enterococalspecies are rarely isolated. Enterococcus faecalis is a main specieswhich is known to have pathogenic factors such as hemolytic toxin andprotease, but basically enterococcal species are thought to be weaklypathogenic.

Enterococci are naturally resistant to cephem antibiotics and many ofthem are resistant to aminoglycoside, macrolide, tetracycline,chloramphenicol, penicillin, lincomycin antibiotics, etc. Since 1980'swhen the third generation cephem antibiotics were introduced, thenosocomial infection caused by multi-resistant enterococci has beenreported. Because enterococci are indigenous microorganisms in theintestine, in most cases, feces are the source of the nosocomialinfection. It is pointed out that hands of health care workers andhospital environment are involved in the infection. If drug resistantenterococci emerge, they are expected to quickly spread to communitiesbecause enterococci are also indigenous in the intestine of many ofdomestic and pet animals and are detected from sewages and theirprocessing facilities.

Vancomycin resistant enterococci (hereinafter referred to as VRE) werereported in the late 1980's in Europe and has quickly spread from the USand Europe to all over the world. People pay great attention to VRE asthe most difficult nosocomial infection organism in treatment. VRE areenterococci that have rendered themselves resistant to vancomycin and,as a result, show resistance to almost all existing antibiotics. Thereare no antibiotics which ensure clinical effectiveness against VREinfection except for those currently being developed. There are someisolated deaths from serious infection such as sepsis. Vancomycinresistant genes are known to be transferred to various Gram positivemicroorganisms through plasmids or chromosomes. People are concernedabout the risk of transferring of vancomycin resistant genes tomethicillin resistant Staphylecoccus aureus (hereinafter referred to asMRSA) to which vancomycin is the only effective drug.

CDC (Centers for Disease Control and Prevention) reported recentincrease in the clinical VRE isolation rate and presented detailedmeasures, stating that each medical institution should establish its owncomprehensive strategy which suits to each facility in order to check,prevent and control VRE infection and settlement. In the report; CDCsays that microorganism testing laboratories are front lines preventingVRE transmission in the hospitals and are required to have the abilityto identify enterococci quickly and precisely and to detect VRE in orderto find VRE infection and settlement. This is to save labor-intensivecontainment which is complex and costly and which will become necessarywhen the detection of VRE infection is delayed. CDC also suggesteddetailed VRE detection tests and emphasized the importance of thedetection tests.

VRE has been also frequently reported in Japanese medical institutionsrecently and has been reported to be detected in meats etc. Thus peopleare concerned about the spread of VRE in Japan. “Infectious diseasecontrol laws” were considerably revised in April, 2000, and “laws forinfection prevention and treatment for patients with infectious disease”were put into effect. In the laws, VRE infection is designated as ClassIV infection, meaning that all the infections have to be reported.Currently, VRE is categorized in six types, Van A VRE, Van B VRE, Van CVRE, Van D VRE, Van E VRE and Van G VRE, depending on resistance genes.These VREs show different resistance patterns against vancomycin andteicoplanin which is a glycopeptide antibiotic and a vancomycinhomologue, depending on their resistance genes. The resistance patternsare shown below:

Van A VRE carries van A gene as a resistance gene, and the minimuminhibitory concentration (hereinafter referred to as MIC) of vancomycinis 64->1000 μg/ml and the MIC of teicoplanin is 16-512 μg/ml.

Van B VRE carries van B gene as a resistance gene, and the MIC ofvancomycin is 4->1000 μg/ml and the MIC of teicoplanin is 0.5-1 μg/ml.

Van C VRE carries van C gene as a resistance gene, and the MIC ofvancomycin is 2-32 μg/ml and the MIC, of teicoplanin is 0.5-1 μg/ml.

Van D VRE carries van D gene as a resistance gene, and the MIC ofvancomycin is 64-1024 μg/ml and the MIC of teicoplanin is 4-1024 μg/ml.

Van E VRE carries van E gene as a resistance gene, and the MIC ofvancomycin is 16 μg/ml and the MIC of teicoplanin is 0.5 μg/ml.

Van G VRE carries van G gene as a resistance gene, and the MIC ofvancomycin is 12-16 μg/ml and the MIC of teicoplanin is 0.5 μg/ml.

Of the above VREs, Van A VRE and Van B VRE are clinically significantbecause the resistance genes thereof can be transmitted to other Grampositive strains through plasmids and chromosomes, and are resistant tomany current antibiotics. In the whole description of the invention inthe specification and claims, “Van A and Van B VRE” means “Van A VRE”and “Van B VRE.” In contrast, Van C VRE is clinically less significantbecause Van C gene is a natural resistance gene which Enterococcuscasseliflavus, Enterococcus gallinarum, Enterococcus flavescens, etcoriginally have, is not transmissible and has relatively low resistanceto various antibiotics.

Van D VRE, Van E VRE and Van G VRE are clinically less significantbecause they have been reported only several times in the whole worldand are not transmissible.

As described above, Van A and Van B VRE are clinically very significantbecause there are no effective treatments for infection thereof, andthey have risks of causing contamination widely in in- and out-hospitalenvironments and of causing serious nosocomial infection. Hence it isrecommended that medical institutions carry out screening tests for VREto detect Van A and Van B VRE. The screening tests should be quick andprecise, too. Currently, the following VRE screening tests are carriedout:

Enterococci constitute a part of the indigenous bacterial flora of theintestine so that the intestine draws attention as a site carrying VRE.Thus for VRE screening tests, fecal specimens and rectal swabs aremainly used. In the case these specimens are used for VRE screeningtests, they are directly inoculated to enterococci selective mediacontaining 6-8 μg/ml of vancomycin, and incubated at 35-37° C. for 24 to48 hours. Colonies grown on the media are tested for identification,vancomycin susceptibility and detection of resistance genes to detectVRE. The enterococci selective media containing 6-8 μg/ml of vancomycinused here are commercially available from several medium manufacturersas the VRE selective medium.

However, VRE selective media currently used for testing allow growth ofclinically less significant Van C VRE as well as clinically significantVan A and Van B VRE.

In addition, it is impossible to differentiate on the media between VanA and Van B VRE, and Van C VRE which grow on the media.

When VRE screening tests are carried out using VRE selective mediacurrently used for testing, additional tests should be carried out todetermine whether grown VRE is clinically significant Van A and Van BVRE or clinically less significant Van C VRE.

Van C VRE is indigenous in the intestine of healthy individuals. Thus ifVRE screening tests are carried out using VRE selective media currentlyavailable and specimens such as feces and the like, numerous Van C VREwould be detected. Accordingly current testing methods becomes complexbecause said additional tests are required.

In addition, it is highly likely that Van A and Van B VRE could bemissed when both of clinically significant Van A and Van B VRE andclinically less significant Van C VRE are present in specimens.

Furthermore, some Van B VRE with a low vancomycin MIC grow very poorlyon currently used VRE selective media. Said strains sometimes do notgrow on said media at all so that if this kind of strains exists inspecimens, it is highly likely that the strains are not detected.

Altogether, if selective media used for VRE screening suppress thegrowth of Van C VRE but support the growth of Van A and Van B VRE alone,they can reduce escapees in detection and improve by leaps and boundsthe complex procedures accompanied with current tests.

SUMMARY OF THE INVENTION

The present inventors took the above into account and intended todevelop Van A and Van B VRE detection media that allow the selectivegrowth of clinically significant Van A and Van B VRE but suppress thegrowth of clinically less significant Van C VRE. We focused on andstudied the vancomycin resistant mechanism of Van A and Van B VRE andfound substances supporting the cell wall synthesis system of Van A andVan B VRE. We successfully made detection media that selectively growclinically significant Van A and Van B VRE but suppress the growth ofclinically less significant Van C VRE by adding the substances to mediaand completed this invention.

The invention provides Van A and Van B VRE detection media thatselectively grow clinically significant Van A and Van B VRE as well as amethod of selectively detecting Van A and Van B VRE from testingmicroorganisms and specimens using said media so that detection miss isreduced and complex testing procedures currently used are improved.

To solve the above problems, the invention described in claim 1 is a VanA and Van B VRE detection medium where vancomycin, D-cycloserine andD-lactate are added to a culture medium formula where enterococci cangrow.

The invention described in claim 2 is a Van A and Van B VRE detectionmedium where 32-256 μg/ml of vancomycin, 1-64 μg/ml of D-cycloserine and0.025-0.8 mol/L of DL-sodium lactate are added to a culture mediumformula where enterococci can grow. The invention described in claim 3is a method of selectively detecting Van A and Van B VRE from testingmicroorganisms and specimens using a Van A and Van B VRE detectionmedium of claims 1 or 2.

The invention described in claim 4 is a method of selectively detectingVan A and Van B VRE, comprising inoculating a suspension of a testingmicroorganism to a Van A and Van B VRE detection medium of claims 1 or2, and incubating the medium at 35-37° C. for 24-48 hours.

The invention described in claim 5 is a method of selectively detectingVan A and Van B VRE, comprising inoculating a specimen to a Van A andVan B VRE detection medium of claims 1 or 2, and incubating the mediumat 35-37° C. for 24-48 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described as follows;

First of all, vancomycin, D-cycloserine and D-lactic acid used in thepresent invention are described.

Vancomycin is discovered in 1954 and introduced into clinical usage in1958. It is a bactericidal glucopeptide antibiotic produced byAmycolatopsis orientalis which has a broad spectrum for Gram positiveorganisms. Since vancomycin has an adverse effect on the kidneys,intestines, etc, it has been used primarily for Gram positive bacteriainfection in limited cases for which β-lactam antibiotics can not beused. Vancomycin has begun to attract attention as a therapeutic agentfor penicillin resistant enterococci infection, pseudomembrane enteritisby C. difficile and MRSA which rapidly spread worldwide in the late1970s, and the drug has become to be used more and more frequently.However, after 1985, Pediococcus and Leuconostoc infections inimmunocompromised patients have been reported in a row. Pediococcus andLeuconostoc strains had been regarded as nonpathogenic lactic acidbacteria which are naturally resistant to vancomycin. People have beenconcerned about the emergence of vancomycin resistant bacteria. Usually,vancomycin hydrochloride is white or light yellow crystalline powder andis used as an oral drug. For the present invention, vancomycinehydrochloride is preferably used.

D-cycloserine is white or light yellowish white crystalline powderderived from Streptomyses garyphalus and is known as an anti-TB drugthat is clinically used for the treatment of pulmonary tuberculosis viaoral administration. D-cycloserine has direct effects on ligase thatsynthesizes D-alanyl-D-alanine which is a part of a cell wallconstituting block of bacterial cells in cell wall construction, andinhibits cell wall construction.

In the present invention, commercially available D-lactic acid can beused without problems. Commercially available lactic acid is usually ina racemic modification or its compound, and is widely used inmanufacturing soft drinks and confectionery as an acidulant or apreservative. For the present invention, DL-sodium lactate, which is aD-lactic acid compound, is preferably used. DL-sodium lactate providesD-lactic acid in constructing D-alanyl-D-lactic acid which is a cellwall constituting block of VRE.

Next theory of the present invention is described. Because thevancomycin MIC of Van C VRE is 2-32 μg/ml, it is assumed that theconcentration of vancomycin to be added to the culture medium should bemore than 32 μg/ml to suppress growth. However some of Van B VRE have alow vancomycin MIC which is less than 32 μg/ml. If more than 32 μg/ml ofvancomycin is simply added to culture medium, Van B VRE with lowvancomycin MIC would be suppressed, resulting in detection miss of saidVan B VRE. Hence, the vancomycin concentration for VRE selective mediacurrently used is 6-8 μg/ml and actually cannot be increased further.

The present inventors contemplated that if the MIC of Van B VRE with lowvancomycin MIC was able to be increased to more than 32 μg/ml, Van A andVan B VRE was able to be differentiated from Van C VRE by the presenceor absence of growth in culture media containing 32 μg/ml of vancomycin.The present inventors paid attention to the resistance mechanism of VanA and Van B VRE. Van A and Van B VRE become vancomycin resistant byreplacing D-alanyl-D-alanine with D-alanyl-D-lactic acid.D-alanyl-D-alanine is a part at the penta-peptide end of the cell walland on which vancomycin has an effect.

Van A VRE, highly resistant to vancomycin, has 8 known vancomycinresistance genes, van R, van S, van H, van A, van X, van Y and van Z.Van H gene produces dehydrogenase and changes pyruvic acid in cells toD-lactic acid. Van A gene binds thus-produced D-lactic acid to D-alaninein cells to produce D-alanyl-D-lactic acid. Thus-producedD-alanyl-D-lactic acid is incorporated into a cell wall component blockto form a cell wall which shows resistance to vancomycin.

D-alanyl-D-alanine, a vancomycin target, is continuously produced byligase which Enterococci inherently possess. Thus-producedD-alanyl-D-alanine is degraded by D, D-dipeptidase produced by van Xgene or D, D-carboxypeptidase produced by van Y gene to be reconstructedinto D-alanyl-D-lactic acid. This series of reactions removesD-alanyl-D-alanine completely and confers Van A VRE high resistance tovancomycin. While Van A VRE shows high resistance to vancomycin, thevancomycin MIC of some of Van B VRE is low. The present inventorsassumed that the reason for Van B VRE having low vancomycin MIC might beattributed to two factors.

One of the factors would be D-alanyl-D-alanine left behind. Van B VREwith low vancomycin MIC would have weak activities in reconstructingD-alanyl-D-lactic acid from D-alanyl-D-alanine produced by ligase whichenterococci inherently possess. Hence D-alanyl-D-alanine, a vancomycintarget, would remain and thereby confer Van B VRE low vancomycin MIC.

Another factor would be a lack in D-lactic acid production. Van B VREwith low vancomycin MIC would not produce a sufficient amount ofD-lactic acid, which would lead to produce an insufficient amount ofD-alanyl-D-lactic acid, and thereby vancomycin MIC lowers relatively.

The present inventors assumed that Van B VRE with low vancomycin MICwould have one of the both factors or both of them. The presentinventors thought that if both factors are concurrently removed, thevancomycin MIC of Van B VRE with low MIC would increase, and tookfollowing measures to remove those factors.

As a measure to remove D-alanyl-D-alanine left behind, factor 1,D-cycloserine is added to culture medium to suppress D-alanyl-D-alanineproduction. As described above, D-cycloserine directly acts on ligasewhich produces D-alanyl-D-alanine (a target of vancmycin), andsuppresses the synthesis thereof. As a result D-alanyl-D-alaninesynthesis itself is suppressed, leading to relative increase inD-alanyl-D-lactic acid production, resulting in the increased vancomycinMIC.

In other words D-cycloserine may work the same way as van X and van Ygenes as mentioned above, to reconstruct D-alanyl-D-lactic acid fromD-alanyl-D-alanine. The present inventors made experiments as follows:

TEST EXAMPLE 1

Measurement of vancomycin MIC changes when D-cycloserine is added Howthe vancomycin MIC of the following enterococcal strains changes wasinvestigated by adding D-cycloserine to culture medium.

Test Strains

In this test, the following strains are used;

Enterococcus faecalis ATCC29212 that does not carry vancomycin 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5 Van A VRE 5 Van B VRE 45 A total of 70 strainsis investigated.

Enterococcus faecalis ATCC29212 that does not carry vancomycinresistance genes.

Test Method

96-well microplates are used for measurement. According to thecheckerboard method, vancomycin ranging from a final concentration of0.5-512 μg/ml was placed in 11 columns and D-cycloserine ranging from afinal concentration of 1-64 μg/ml was placed in 7 rows.

Brain heart infusion broth was used as a culture medium. Eachentrococcal strain was aerobically incubated in brain heart infusionbroth at 35° C. for 48 hours and diluted 100-fold with brain heartinfusion broth. One hundred μl of the diluted suspension was inoculatedin each well. After the microplate was incubated at 35° C. for 18 hours,the vancomycin MIC was determined and the result was shown in Table 1.

Test Results

The addition of D-cycloserine did not change the vancomycin MIC in thefollowing strains and the vancomycin MIC was less than 2 μg/ml.

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes The addition of D-cycloserine did not change thevancomycin MIC in the following strains and the vancomycin MIC was lessthan 16 μg/ml. Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5

In contrast, the addition of D-cycloserine increased the vancomycin MICor maintained the vancomycin MIC as high as more than 512 μg/ml in allof the following strains.

Van A VRE 5 Van B VRE 45Discussion

The addition of D-cycloserine increased the vancomycin MIC of Van B VREwith low vancomycin MIC. The vancomycin MIC was increased by theaddition of D-cycloserine in the concentration ranging from 1 μg/ml to64 μg/ml and the optimum concentration of addition was confirmed to beat 32 μg/ml.

In the next step, DL-sodium lactate, which is a D-lactic acid compound,was added to culture medium to provide sufficient D-lactic acid forproducing D-alanyl-D-lactic acid as a means to supplement aninsufficient production of D-lactic acid. The present inventorsanticipated that the addition of DL-sodium lactate would produce asufficient amount of D-alanyl-D-lactic acid, which leads to the increaseof D-alanyl-D-lactic acid production relatively, thereby increasing thevancomycin MIC. D-lactic acid presumably has a similar effect asD-lactic acid production by van H gene which was mentioned above. Thefollowing study was conducted.

TEST EXAMPLE 2

Measurement of vancomycin MIC changes when D-lactic acid is added.

Changes of the vancomycin MIC of following enterococcal strains wereinvestigated by adding DL-sodium lactate.

Test Strains

In this study, the following strains are used;

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5 Van A VRE 5 Van B VRE 45 A total of 70 strainsis investigated.Test Method

96-well microplates are used for measurement. According to thecheckerboard method, vancomycin ranging from a final concentration of0.5-512 μg/ml was placed in 11 columns and DL-sodium lactate rangingfrom a final concentration of 0.025-1.6 mol/l was placed in 7 rows.

Brain heart infusion broth was used as a culture medium. Eachentrococcal strain was aerobically incubated in brain heart infusionbroth at 35° C. for 48 hours and diluted 100-fold with brain heartinfusion broth. One hundred μl of the diluted suspension was inoculatedin each well. After the microplate was incubated at 35° C. for 48 hours,the vancomycin MIC was determined and the result was shown in Table 2.

Test Results

The addition of DL-sodium lactate did not change the vancomycin MIC inthe following strains and the vancomycin MIC was less than 2 μg/ml.

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes The addition of DL-sodium lactate did not change thevancomycin MIC in the following strains, either and the vancomycin MICwas less than 16 μg/ml. Van C type, Enterococcus casseliflavus 5 Van Ctype, Enterococcus gallinarum 5

In contrast, the addition of DL-sodium lactate increased the vancomycinMIC or maintained the vancomycin MIC as high as more than 512 μg/ml inall of the following strains.

Van A VRE 5 Van B VRE 45Discussion

It was confirmed that the addition of DL-sodium lactate increased thevancomycin MIC of Van B VRE with low vancomycin MIC. The vancomycin MICwas increased by the addition of DL-sodium lactate in the concentrationranging from 0.025 to 0.8 mol/l.

However the organisms tend to grow worse in line with the increase ofthe added amount at the concentration of more than 0.4 mol/l, theoptimum concentration of addition was concluded to be at 0.2 mol/l.

From the result of studies 1 and 2, it was confirmed that enterococcalstrains not carrying vancomycin resistance genes as well as Van C VREdid not change the vancomycin MIC by adding D-cycloserine or DL-sodiumlactate, and their MICs were all less than 32 μg/ml.

On the other hand, it was confirmed that Van A and Van B VRE increasedtheir MIC in all strains by adding D-cycloserine or DL-sodium lactateand their MIC was more than 32 μg/ml.

It is expected that enterococcal strains not carrying vancomycinresistance genes as well as Van C VRE do not grow in culture mediumcontaining more than 32 μg/ml of vancomycin even when the medium isspiked with D-cycloserine and DL-sodium lactate. It is also expectedthat Van A and Van B VRE grow even in culture medium containing morethan 32 μg/ml of vancomycin by adding D-cycloserine and DL-sodiumlactate to culture medium. The following studies are conducted.

TEST EXAMPLE 3

Measurement of the growth of VRE in culture medium where D-cycloserineand D-lactic acid are added.

Growth of various enterococcal strains was investigated in culturemedium containing D-cycloserine, DL-sodium lactate and vancomycin bydilution-culture test method.

Test Strains

In this study, the following strains are tested;

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5 Van A VRE 5 Van B VRE 45 A total of 70enterococcal strains is investigated.Test Method

Culture media used are as follows;

-   1.5% sheep blood agar-   2. BHI agar containing 32 μg/ml of vancomycin-   3. BHI agar containing 32 μg/ml of vancomycin, 32 μg/ml of    D-cycloserine and 0.2 mol/l of DL-sodium lactate

Solutions of the above enterococcal strains were prepared to haveturbidness of No. 5 MacFarland and the undiluted solution was used tomake 10⁻¹ to 10⁻⁶ dilutions. Ten μl of each of these dilutions werespot-inoculated to each of the above three culture media and aerobicallyincubated at 35° C. for 48 hours. Colonies grown were counted andcompared to the growth on 5% sheep blood agar, which is a control. Theresults are shown in Table 3, 4 and 5.

In the Tables “+”, “++”, “+++” and “(+)” mean the following.

+++: more than 10,000 colonies were grown. ++: 1,001 to 10,000 colonieswere grown. +: 500 to 1,000 colonies were grown. (+): colonies wereobserved, but they were very small.

The above symbols mean the same in Tables 6-8.

Test Results

On BHI agar containing 32 μg/ml of vancomycin as well as BHI agarcontaining 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, and 0.2mol/l of DL-sodium lactate, the following strains did not grow at all;

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5

On the contrary, all strains of Van A VRE (5 strains) grew as well onboth of the BHI agar containing 32 μg/ml of vancomycin and thatcontaining 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine and 0.2mol/l of DL-sodium lactate as on 5% sheep blood agar.

For Van B VRE (45 strains), one strain did not grow at all and 12strains grew extremely poorly on BHI agar containing 32 μg/ml ofvancomycin compared to on 5% sheep blood agar. It was considered thatthese strains were suppressed to grow in the medium containing 32 μg/mlof vancomycin because their vancomycin MIC was low.

Whereas all of the Van B VRE strains grew as well on BHI agar containing32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine and 0.2 mol/l ofDL-sodium lactate as on 5% sheep blood agar. It was considered that VanB VRE with low vancomycin MIC was increased in its vancomycin MIC byadding D-cycloserine and DL-sodium lactate to culture medium so that VanB VRE was able to grow in culture medium containing 32 μg/ml ofvancomycin.

Discussion

From the above test results, enterococcal strains not carryingvancomycine resistance genes as well as Van C VRE can be clearlydifferentiated from Van A and Van B VRE by the presence or absence ofgrowth on BHI agar spiked with 32 μg/ml of vancomycin, 32 μg/ml ofD-cycloserine and 0.2 mol/l of DL-sodium lactate.

The present inventors attempted to produce Van A and Van B VRE detectionmedia based on the above studies. The concentration of vancomycin ispreferably 32-256 μg/ml and, further, it was concluded that its optimumconcentration is 32 μg/ml. The concentration of D-cycloserine ispreferably 1-64 μg/ml and, further, it was concluded that its optimumconcentration is 32 μg/ml. The concentration of DL-sodium lactate ispreferably 0.025-0.8 mol/l and, further, it was concluded that itsoptimum concentration is 0.2 mol/l.

BHI agar spiked with 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2μg/ml of amphotericin B and 50 μg/ml of 2,3,5-triphenyltetrazoliumchloride (hereinafter referred to as TTC) was used as a medium where VREcan grow. Polymyxin B and aztreonam are added in order to suppressgrowth of Gram negative bacilli and amphotericin B to suppresses yeast,and TTC is added as a color agent for visualization of VRE colonies. BHIagar containing 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/mlof amphotericin B and 50 μg/ml of TTC is only one of the medium exampleswhere enterococci can grow and is not the essential structural featureof the invention. Any culture media containing ingredient for the growthof enterococci can be used in the invention.

The present inventors have successfully produced Van A and Van B VREdetection media including a representative example, namely, BHI agarspiked with 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/lof DL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2μg/ml of amphotericin B and 50 μg/ml of TTC.

The present inventors attempted to selectively detect Van A and Van BVRE using a representative example, BHI agar containing 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate,20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericinB and 50 μg/ml of TTC. Specifically, Van A and Van B VRE are selectivelydetected by the following testing procedure.

In a concrete testing procedure when bacteria suspected of Van A and VanB VRE are detected from clinical specimens, the specimens are suspended,inoculated into the Van A and Van B VRE detection media of the presentinvention, and incubated at 35-37° C. for 24-48 hours. Van A and Van BVRE are selectively detected by the presence or absence of growth.

Alternatively, in another testing procedure where Van A and Van B VREare directly detected from specimens, specimens are inoculated into theVan A and Van B VRE detection media of the present invention directlyand incubated at 35-37° C. for 24-48 hours. Van A and Van B VRE areselectively detected by the presence or absence of growth.

EXAMPLES

Specific procedures are further described in detail in examples.

Example 1

Van A and Van B VRE are selectively detected by inoculating a suspensionof testing bacteria into medium

Two media, Enterococcosel agar with 8 μg/ml of vancomycin, which is usedin the current test method, and BHI agar containing 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate,20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericinB and 50 μg/ml of TTC were used for testing growth of variousenterococal strains on dilution media by dilution-culture test method.

Test Strains

In this study, the following strains are tested;

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes Van C type, Enterococcus casseliflavus 5 Van C type,Enterococcus gallinarum 5 Van A VRE 5 Van B VRE 45 A total of 70 strainsis investigated.Test Method

Three media, agar with 5% sheep blood as a control medium,Enterococcosel agar with 8 μg/ml of vancomycin, and BHI agar of thepresent invention spiked with 32 μg/ml of vancomycin, 32 μg/ml ofD-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml of polymyxin B,20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTCwere used.

Solutions of the above enterococcal strains were prepared to haveturbidness of No. 5 MacFarland and the undiluted solution was used tomake 10⁻¹ to 10⁻⁶ dilutions. Ten μl of each of these dilutions werespot-inoculated to each of the above three culture media and aerobicallyincubated at 35 C for 48 hours. Colonies grown were counted and comparedto the growth on 5% sheep blood agar, which is a control. The resultsare shown in Tables 6, 7 and 8.

Test Results

On Enterococcosel agar with 8 μg/ml of vancomycin and BHI agar spikedwith 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l ofDL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2μg/ml of amphotericin B and 50 μg/ml of TTC, the following strains didnot grow at all;

Enterococcus faecalis ATCC29212 that does not carry vancomycine 1resistance genes Enterococcus faecalis that does not carry vancomycineresistance 3 genes Enterococcus faecium that does not carry vancomycineresistance 3 genes Enterococcus avium that does not carry vancomycineresistance 3 genes

Van C Enterococcus casseliflavus (5 strains) and Van C Enterococcusgallinarum (5 strains) were all grown on Enterococcosel agar with 8μg/ml of vancomycin but were not grown at all on BHI agar spiked with 32μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodiumlactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml ofamphotericin B and 50 μg/ml of TTC.

Van A VRE (5 strains) grew well on Enterococcosel agar with 8 μg/ml ofvancomycin and BHI agar spiked with 32 μg/ml of vancomycin, 32 μg/ml ofD-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml of polymyxin B,20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTC tothe same extent as on agar with 5% sheep blood.

Eight strains of Van B VRE (45 strains) in higher dilution grew poorlyon Enterococcosel agar with 8 μg/ml of vancomycin but grew well on BHIagar spiked with 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2mol/l of DL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml ofaztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTC to the sameextent as on agar with 5% sheep blood.

Discussion

According to the results of Example 1, it is confirmed enterococcalstrains not carrying vancomycine resistance genes as well as Van C VREcan be clearly differentiated from Van A and Van B VRE by the presenceor absence of growth on BHI agar spiked with 32 μg/ml of vancomycin, 32μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml ofpolymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50μg/ml of TTC. In summary, when Van A and Van B VRE are detected fromtesting bacteria, they are possible to be selectively detected by thepresence or absence of growth on the media after bacteria weresuspended, inoculated to Van A and Van B VRE detection media developedby the present inventors, represented by BHI agar spiked with 32 μg/mlof vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodiumlactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml ofamphotericin B and 50 μg/ml of TTC, and incubated for 24-48 hours at 35°C.-37° C.

Example 2

Van A and Van B VRE are selectively detected by directly inoculatingspecimens to media

Two media, Enterococcosel agar with 8 μg/ml of vancomycin, which is usedin the current test method, and BHI agar spiked with 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate,20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericinB and 50 μg/ml of TTC were used for screening VRE from fecal specimensof patients and compared.

For testing media, 49 fecal specimens of patients were used in thisExample.

Test Methods

About an equal amount of sterile physiological saline was added to eachof fecal specimens of patients and mixed well. A loopful of resultantpaste samples was inoculated to each medium. After 48 hours ofincubation at 35° C., colonies grown on plates were carefully examined.Results are shown in Table 9.

Test Results

In 13 specimens of 49 specimens (26.5%), 16 Van C VRE strains weredetected on Enterococcosel agar with 8 μg/ml of vancomycin. Conversely,Van C VRE was not detected at all on BHI agar spiked with 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate,20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericinB and 50 μg/ml of TTC. Van A and Van B VRE were not detected in anymedia in this screening test.

Discussion

According to the results of Example 2, it was confirmed that also inactual fecal specimens of patients the growth of Van C VRE wassuppressed on Van A and Van B VRE detection media, represented by BHIagar containing 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2mol/l of DL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml ofaztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTC.

Known Van B VRE was added to fecal specimens of patients since Van A andVan B VRE were not detected in the screening test of Example 2, and thespecimens were tested by the following test to see how many of the addedVan B VRE could be detected.

Example 4

Test for confirming the detection rate of Van B VRE from Van B VREinoculated specimens

Testing specimens were 2 patient specimens, Sample 1 and 2, in which VanA and Van B VRE were confirmed not to be present by prior testing, andeach of known 3 Van B VRE strains was inoculated to each sample. Theinoculating amount of Van B VRE was adjusted to 10-20 cells per 10 μl.

Test Methods

Two media, Enterococcosel agar with 8 μg/ml of vancomycin currently usedfor a test, and BHI agar spiked with 32 μg/ml of vancomycin, 32 μg/ml ofD-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml of polymyxin B,20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTCwere used. Ten μl of specimens spiked with Van B VRE were inoculated toeach medium. After 48 hours of incubation at 35° C., colonies grown onplates were carefully examined. Results are shown in Table 10.

Test Results

In a patient fecal specimen Sample 1, about 10⁷ cells of Van C VRE werepresent in 10 μl. Van C VRE grew on Enterococcosel agar with 8 μg/ml ofvancomycin currently used for a test, which resulted in failing todetect all three strains of Van B VRE. In contrast, Van C VRE wascompletely suppressed on BHI agar spiked with 32 μg/ml of vancomycin, 32μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml ofpolymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50μg/ml of TTC. It was confirmed that almost same number of cells wasdetected from the media as that which were inoculated.

Van C VRE did not grow on the media to which Sample 2, a patient fecalspecimen, was inoculated. One of three Van B VRE strains was notdetected at all on Enterococcosel agar with 8 μg/ml of vancomycincurrently used for a test.

Discussion

It was revealed from Sample 1 that Enterococcosel agar with 8 μg/ml ofvancomycin currently used for a test might fail to detect Van A and VanB VRE in case specimens contained numerous Van C VRE.

In contrast, it was confirmed that because Van C VRE was completelysuppressed, the target bacteria, Van A and Van B VRE, were able to bedetected on Van A and Van B VRE detection media of the presentinvention, even if few of them existed, represented by BHI agar spikedwith 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l ofDL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2μg/ml of amphotericin B and 50 μg/ml of TTC.

It was evident from Sample 2 that there was Van B VRE which was notdetected on Enterococcosel agar with 8 μg/ml of vancomycin currentlyused for a test when the number of cells was few. In contrast, such VanB VRE grew well and was detected, even if there were few in specimens,on newly developed BHI agar spiked with 32 μg/ml of vancomycin, 32 μg/mlof D-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml of polymyxinB, 20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50 μg/ml of TTC.Altogether, it is demonstrated from the Test Examples and Examples thatVan A and Van B VRE detection media, specifically, represented by BHIagar of the present invention containing 32 μg/ml of vancomycin, 32μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate, 20 μg/ml ofpolymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericin B and 50μg/ml of TTC can selectively detect clinically important Van A and Van BVRE.

As described above, in a concrete testing procedure when bacteriasuspected of Van A and Van B VRE are detected from clinical specimens,the specimens are suspended, inoculated into the Van A and Van B VREdetection media of the present invention, and incubated at 35-37° C. for24-48 hours. After that it is possible to selectively detect Van A andVan B VRE by the presence or absence of growth.

Alternatively, in another testing procedure where Van A and Van B VREare directly detected from specimens, specimens are inoculated into theVan A and Van B VRE detection media of the present invention andincubated at 35-37° C. for 24-48 hours. It is possible to selectivelydetect Van A and Van B VRE by the presence or absence of growth.

INDUSTRIAL APPLICABILITY

Clinically important Van A and Van B VRE are selectively detected fromtesting microorganisms or specimens using Van A and Van B VRE detectionmedia of the present invention, specifically, represented by BHI agarspiked with 32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/lof DL-sodium lactate, 20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2μg/ml of amphotericin B and 50 μg/ml of TTC. The method for selectivelydetecting Van A and Van B VRE which was developed newly is markedlysimple and increases detection accuracy compared to current detectionmethods.

Van A and Van B VRE detection media of the present invention and amethod of selectively detecting Van A and Van B VRE using the media arevery useful in the present medical field where early detection andprevention of infection of Van A and Van B VRE are strongly sought.

TABLE 1 Changes in vancomycin MIC values by adding D-cycloserin(incubated at 35° C. for 48 hours) vancomycin MIC values(μg/ml)Resistance Amount of D-cycloserin added (μg/ml) Seq. Organism # Strainsgene 0 1 2 4 8 16 32 64 1 E. faecalis ATCC 29212 E. faecalis None 2 2 22 2 2 2 2 2 E. faecalis Control 1 E. faecalis None 2 2 2 2 2 2 2 2 3 E.faecalis Control 2 E. faecalis None 2 2 2 2 2 2 2 2 4 E. faecalisControl 3 E. faecalis None 2 2 2 2 2 2 2 2 5 E. faecium Control 1 E.faecium None 2 2 1 1 1 1 1 0.5 6 E. faecium Control 2 E. faecium None 11 1 1 1 1 1 0.5 7 E. faecium Control 3 E. faecium None 2 2 2 1 2 1 1 — 8E. avium Control 1 E. avium None 1 1 1 1 1 1 1 — 9 E. avium Control 2 E.avium None 2 1 2 1 2 2 1 — 10 E. avium Control 3 E. avium None 1 1 1 1 11 1 0.5 11 E. casseliflavus Control 1 E. casseliflavus vanC 8 8 8 8 8 88 8 12 E. casseliflavus Control 2 E. casseliflavus vanC 8 8 8 8 8 8 8 813 E. casseliflavus Control 3 E. casseliflavus vanC 8 8 8 8 8 8 8 8 14E. casseliflavus Control 4 E. casseliflavus vanC 8 8 8 8 8 8 8 8 15 E.casseliflavus Control 5 E. casseliflavus vanC 8 8 8 8 8 8 8 8 16 E.gallinarum Control 1 E. gallinarum vanC 16 16 16 16 16 16 16 16 17 E.gallinarum Control 2 E. gallinarum vanC 16 16 16 16 16 16 16 16 18 E.gallinarum Control 3 E. gallinarum vanC 16 16 16 16 16 16 16 16 19 E.gallinarum Control 4 E. gallinarum vanC 16 16 16 16 16 16 16 16 20 E.gallinarum Control 5 E. gallinarum vanC 16 16 16 16 16 16 16 16 21 TW3491 E. faecium vanA >512 >512 >512 >512 >512 >512 >512 >512 22 TW 4558E. faecium vanA 256 256 256 256 512 512 512 — 23 TW 4559 E. faecium vanA512 >512 >512 >512 512 512 512 512 24 TW 4561 E. faecium vanA 512 512512 512 512 512 >512 512 25 TW 4590 E. faeciumvanA >512 >512 >512 >512 >512 >512 >512 >512 26 TW 3492 E. faecium vanB32 32 32 32 64 64 128 128 27 TW 4560 E. faecium vanB 64 64 64 64 64 128128 128 28 TW 4589 E. faecalis vanB 16 16 16 16 16 32 64 64 29 TW 5246E. faecalis vanB 256 512 512 512 512 512 512 >512 30 TW 5247 E. faeciumvanB 16 64 64 128 128 256 256 2 31 TW 5604 E. faecium vanB 8 8 16 64 6464 64 — 32 TW 5607 E. faecium vanB 512 512 512 512 512 512 512 512 33 TW5608 E. faecalis vanB >512 >512 >512 >512 >512 >512 >512 >512 34 TW 5609E. faecalis vanB 128 256 256 256 256 256 256 256 35 TW 5610 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 >512 36 TW 5611 E. faecalis vanB16 16 16 32 32 64 64 64 37 TW 5645 E. faeciumvanB >512 >512 >512 >512 >512 >512 >512 >512 38 TW 5646 E. faecium vanB512 512 512 512 512 512 512 512 39 TW 5668 E. faecium vanB 32 16 32 3232 32 64 64 40 TW 5669 E. faecium vanB 32 32 32 32 64 64 128 128 41 TW5670 E. faecium vanB 32 32 64 64 64 64 64 64 42 TW 5671 E. faecium vanB16 32 32 32 32 64 64 64 43 TW 5672 E. faeciumvanB >512 >512 >512 >512 >512 >512 >512 >512 44 TW 5682 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 >512 45 TW 5683 E. faecalis vanB128 128 128 128 128 256 256 256 46 TW 5684 E. faecalis vanB 128 256 256256 256 256 256 256 47 TW 5685 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 >512 48 TW 5686 E. faecalis vanB512 512 512 512 512 512 512 512 49 TW 5687 E. faecalis vanB 64 64 64 128128 128 256 256 50 TW 5688 E. faecium vanB 64 64 64 64 64 64 64 64 51 TW5689 E. faecium vanB 128 128 128 256 256 256 256 128 52 TW 5690 E.faecium vanB >512 >512 >512 >512 >512 >512 >512 >512 53 TW 6169 E.faecalis vanB 128 128 128 128 128 128 128 128 54 TW 6170 E. faecalisvanB 16 16 16 16 16 16 32 32 55 TW 6171 E. faecium vanB 16 16 16 16 1616 32 16 56 TW 6172 E. faecium vanB 16 16 16 16 16 16 32 16 57 TW 6173E. faecium vanB 256 256 256 512 512 >512 >512 >512 58 TW 6174 E. faeciumvanB 64 64 64 64 64 128 128 128 59 TW 6175 E. faecium vanB 256 128 256256 256 256 256 128 60 TW 6176 E. faecium vanB 256 256 256 256 256512 >512 >512 61 TW 6177 E. faecalis vanB 256 256 256 256 256 256512 >512 62 TW 6178 E. faecium vanB 256 256 256 256 256 256 256 256 63TW 6179 E. faecalis vanB 32 32 32 32 32 64 64 64 64 TW 6180 E. faeciumvanB >512 >512 >512 >512 >512 >512 >512 >512 65 TW 6181 E. faeciumvanB >512 >512 >512 >512 >512 >512 >512 >512 66 TW 6183 E. faecalis vanB256 256 256 256 256 256 256 128 67 TW 6184 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 >512 68 TW 6185 E. faecium vanB128 128 256 256 256 256 512 >512 69 TW 7515 E. faecalis vanB 64 64 128128 128 128 256 256 70 TW 7516 E. faecalis vanB 512 512 512 512512 >512 >512 >512

TABLE 2 Changes in vancomycin MIC values by adding DL-sodium lactate(incubated at 35° C. for 48 hours) vancomycin MIC values(μg/ml)Resistance Amount of DL-Sodium lactate added (mol/l) Sec. Organism #Strains gene 0 0.025 0.05 0.1 0.2 0.4 0.8 1.6 1 E. faecalis ATCC 29212E. faecalis None 2 2 2 2 2 2 2 1 2 E. faecalis Control 1 E. faecalisNone 1 2 2 2 2 2 2 — 3 E. faecalis Control 2 E. faecalis None 2 2 2 2 22 2 0.5 4 E. faecalis Control 3 E. faecalis None 1 2 2 2 2 2 2 1 5 E.faecium Control 1 E. faecium None 1 1 1 2 2 2 2 1 6 E. faecium Control 2E. faecium None 1 1 1 2 2 2 2 2 7 E. faecium Control 3 E. faecium None 11 2 2 2 2 2 2 8 E. avium Control 1 E. avium None 1 1 1 1 2 2 2 — 9 E.avium Control 2 E. avium None 2 1 2 2 2 2 2 — 10 E. avium Control 3 E.avium None 1 1 1 2 2 2 2 — 11 E. casseliflavus Control 1 E.casseliflavus vanC 8 8 8 8 16 16 16 — 12 E. casseliflavus Control 2 E.casseliflavus vanC 8 8 8 8 16 16 16 — 13 E. casseliflavus Control 3 E.casseliflavus vanC 8 8 8 16 16 16 16 — 14 E. casseliflavus Control 4 E.casseliflavus vanC 8 8 8 8 16 16 16 — 15 E. casseliflavus Control 5 E.casseliflavus vanC 8 8 8 8 16 16 16 — 16 E. gallinarum Control 1 E.gallinarum vanC 8 8 8 8 16 16 16 — 17 E. gallinarum Control 2 E.gallinarum vanC 8 8 16 16 16 16 16 — 18 E. gallinarum Control 3 E.gallinarum vanC 16 8 16 16 16 16 16 — 19 E. gallinarum Control 4 E.gallinarum vanC 8 8 8 16 16 16 16 — 20 E. gallinarum Control 5 E.gallinarum vanC 8 8 8 8 16 16 16 — 21 TW 3491 E. faeciumvanA >512 >512 >512 >512 >512 >512 >512 >512 22 TW 4558 E. faecium vanA256 256 512 512 512 512 >512 16 23 TW 4559 E. faeciumvanA >512 >512 >512 >512 >512 >512 >512 — 24 TW 4561 E. faecium vanA 512512 512 512 >512 >512 >512 — 25 TW 4590 E. faecium vanA512 >512 >512 >512 >512 >512 >512 — 26 TW 3492 E. faecium vanB 32 64 64128 256 512 256 — 27 TW 4560 E. faecium vanB 32 64 128 128 256 >512 >512— 28 TW 4589 E. faecalis vanB 16 256 512 >512 >512 >512 >512 — 29 TW5246 E. faecalis vanB 256 512 512 512 >512 >512 >512 — 30 TW 5247 E.faecium vanB 16 64 128 128 256 >512 >512 64 31 TW 5604 E. faecium vanB 832 64 64 128 512 >512 — 32 TW 5607 E. faecium vanB 512 512 512512 >512 >512 >512 — 33 TW 5608 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 — 34 TW 5609 E. faecalis vanB128 512 512 512 >512 >512 >512 — 35 TW 5610 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 — 36 TW 5611 E. faecalis vanB 1664 64 128 256 512 512 — 37 TW 5645 E. faecium vanB512 >512 >512 >512 >512 >512 >512 — 38 TW 5646 E. faecium vanB 256 512512 >512 >512 >512 >512 — 39 TW 5668 E. faecium vanB 16 32 32 64 64 128128 — 40 TW 5669 E. faecium vanB 64 64 64 128 256 256 512 — 41 TW 5670E. faecium vanB 32 32 32 64 64 128 512 — 42 TW 5671 E. faecium vanB 1616 16 32 64 128 256 — 43 TW 5672 E. faeciumvanB >512 >512 >512 >512 >512 >512 >512 — 44 TW 5682 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 — 45 TW 5683 E. faecalis vanB 64256 512 512 >512 >512 >512 — 46 TW 5684 E. faecalis vanB 128 512 512512 >512 >512 >512 — 47 TW 5685 E. faecalis vanB512 >512 >512 >512 >512 >512 >512 — 48 TW 5686 E. faecalis vanB 512 512512 >512 >512 >512 >512 — 49 TW 5687 E. faecalis vanB 128512 >512 >512 >512 >512 >512 — 50 TW 5688 E. faecium vanB 64 64 64 128256 512 512 — 51 TW 5689 E. faecium vanB 128 128 256 256 512 >512 >512 —52 TW 5690 E. faecium vanB >512 >512 >512 >512 >512 >512 >512 — 53 TW6169 E. faecalis vanB 64 128 256 256 256 >512 >512 — 54 TW 6170 E.faecalis vanB 16 32 64 64 128 128 256 — 55 TW 6171 E. faecium vanB 8 1616 32 64 128 256 — 56 TW 6172 E. faecium vanB 8 16 32 32 64 128 128 — 57TW 6173 E. faecium vanB 512 512 >512 >512 >512 >512 >512 — 58 TW 6174 E.faecium vanB 64 64 128 256 256 >512 >512 — 59 TW 6175 E. faecium vanB128 128 256 256 256 512 >512 — 60 TW 6176 E. faecium vanB 256 256 512512 >512 >512 >512 — 61 TW 6177 E. faecalis vanB 256 256 256512 >512 >512 >512 — 62 TW 6178 E. faecium vanB 256 512 512512 >512 >512 >512 — 63 TW 6179 E. faecalis vanB 32 64 64 64 128 256 512— 64 TW 6180 E. faecium vanB >512 >512 >512 >512 >512 >512 >512 — 65 TW6181 E. faecium vanB >512 >512 >512 >512 >512 >512 >512 — 66 TW 6183 E.faecalis vanB 128 256 512 512 >512 >512 >512 — 67 TW 6184 E. faecalisvanB >512 >512 >512 >512 >512 >512 >512 — 68 TW 6185 E. faecium vanB 128128 128 128 128 >512 >512 — 69 TW 7515 E. faecalis vanB 64 256 512512 >512 >512 >512 — 70 TW 7516 E. faecalis vanB256 >512 >512 >512 >512 >512 >512 —

TABLE 3 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) 5% sheep blood agar DilutionResistance Undiluted Seq. Organism # Strains gene solution ×10⁻¹ ×10⁻²×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC 29212 E. faecalis None ++++++ ++ + 15 0 0 2 E. faecalis Control 1 E. faecalis None +++ +++ ++ + 333 0 3 E. faecalis Control 2 E. faecalis None +++ +++ ++ + 21 1 0 4 E.faecalis Control 3 E. faecalis None +++ +++ ++ + 31 5 0 5 E. faeciumControl 1 E. faecium None +++ +++ ++ + 42 5 0 6 E. faecium Control 2 E.faecium None +++ +++ ++ + 30 1 0 7 E. faecium Control 3 E. faecium None+++ +++ ++ + 25 1 0 8 E. avium Control 1 E. avium None +++ +++ ++ + 22 20 9 E. avium Control 2 E. avium None +++ +++ ++ + 31 2 0 10 E. aviumControl 3 E. avium None +++ +++ ++ + 26 1 0 11 E. casseliflavus Control1 E. casseliflavus vanC +++ +++ ++ + 40 5 0 12 E. casseliflavus Control2 E. casseliflavus vanC +++ +++ ++ + 36 2 0 13 E. casseliflavus Control3 E. casseliflavus vanC +++ +++ ++ + 32 2 0 14 E. casseliflavus Control4 E. casseliflavus vanC +++ +++ ++ + 51 4 0 15 E. casseliflavus Control5 E. casseliflavus vanC +++ +++ ++ + 46 3 0 16 E. gallinarum Control 1E. gallinarum vanC +++ +++ ++ + 28 1 0 17 E. gallinarum Control 2 E.gallinarum vanC +++ +++ ++ + 41 5 0 18 E. gallinarum Control 3 E.gallinarum vanC +++ +++ ++ + 47 5 0 19 E. gallinarum Control 4 E.gallinarum vanC +++ +++ ++ + 50 5 0 20 E. gallinarum Control 5 E.gallinarum vanC +++ +++ ++ + 38 2 0 21 TW 3491 E. faecium vanA +++ +++++ + 58 7 0 22 TW 4558 E. faecium vanA +++ +++ ++ + 68 7 0 23 TW 4559 E.faecium vanA +++ +++ ++ + 59 7 0 24 TW 4561 E. faecium vanA +++ +++ ++ +31 6 0 25 TW 4590 E. faecium vanA +++ +++ ++ + 66 6 0 26 TW 3492 E.faecium vanB +++ +++ ++ + 74 4 2 27 TW 4560 E. faecium vanB +++ +++ ++ +29 3 0 28 TW 4589 E. faecalis vanB +++ +++ ++ + 34 2 1 29 TW 5246 E.faecalis vanB +++ +++ ++ + 39 4 0 30 TW 5247 E. faecium vanB +++ +++++ + 34 4 0 31 TW 5604 E. faecium vanB +++ +++ ++ + 71 10 1 32 TW 5607E. faecium vanB +++ +++ ++ + 28 1 0 33 TW 5608 E. faecalis vanB +++ +++++ + 13 2 0 34 TW 5609 E. faecalis vanB +++ +++ ++ + 30 5 0 35 TW 5610E. faecalis vanB +++ ++ + 34  7 0 0 36 TW 5611 E. faecalis vanB +++ +++++ + 26 4 0 37 TW 5645 E. faecium vanB +++ +++ ++ + 38 5 0 38 TW 5646 E.faecium vanB +++ +++ ++ + 22 3 0 39 TW 5668 E. faecium vanB +++ +++ ++ +66 5 0 40 TW 5669 E. faecium vanB +++ +++ ++ + 71 3 1 41 TW 5670 E.faecium vanB +++ +++ ++ + 47 5 0 42 TW 5671 E. faecium vanB +++ +++ ++++ + 11 0 43 TW 5672 E. faecium vanB +++ +++ ++ + 39 9 5 44 TW 5682 E.faecalis vanB +++ +++ +++ ++ + 16 3 45 TW 5683 E. faecalis vanB +++ +++++ + 54 8 1 46 TW 5684 E. faecalis vanB +++ +++ +++ ++ + 16 3 47 TW 5685E. faecalis vanB +++ +++ ++ + 22 5 0 48 TW 5686 E. faecalis vanB +++ ++++++ ++ + 14 4 49 TW 5687 E. faecalis vanB +++ +++ ++ + 28 3 0 50 TW 5688E. faecium vanB +++ +++ ++ + 49 11 1 51 TW 5689 E. faecium vanB +++ +++++ + 40 1 1 52 TW 5690 E. faecium vanB +++ +++ +++ ++ + 15 2 53 TW 6169E. faecalis vanB +++ +++ +++ ++ + 23 16 54 TW 6170 E. faecalis vanB ++++++ ++ + 45 6 1 55 TW 6171 E. faecium vanB +++ +++ +++ ++ + 8 1 56 TW6172 E. faecium vanB +++ +++ ++ + 30 8 1 57 TW 6173 E. faecium vanB ++++++ ++ ++ + 7 2 58 TW 6174 E. faecium vanB +++ +++ ++ + 58 6 0 59 TW6175 E. faecium vanB +++ +++ ++ + 51 3 0 60 TW 6176 E. faecium vanB ++++++ ++ + 42 9 3 61 TW 6177 E. faecalis vanB +++ +++ ++ + 52 15 2 62 TW6178 E. faecium vanB +++ +++ ++ + 25 6 1 63 TW 6179 E. faecalis vanB ++++++ ++ + 53 18 4 64 TW 6180 E. faecium vanB +++ +++ ++ + 52 4 1 65 TW6181 E. faecium vanB +++ +++ ++ + 30 4 0 66 TW 6183 E. faecalis vanB ++++++ ++ + 16 5 2 67 TW 6184 E. faecalis vanB +++ +++ ++ + 58 13 3 68 TW6185 E. faecium vanB +++ +++ ++ + 60 12 3 69 TW 7515 E. faecalis vanB+++ +++ +++ ++ + 12 1 70 TW 7516 E. faecalis vanB +++ +++ ++ + 47 5 2

TABLE 4 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) BHI agar with 32 μg/ml ofvancomycin Dilution Resistance Undiluted Seq. Organism # Strains genesolution ×10⁻¹ ×10⁻² ×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC 29212 E.faecalis None 0 0 0 0 0 0 0 2 E. faecalis Control 1 E. faecalis None 0 00 0 0 0 0 3 E. faecalis Control 2 E. faecalis None 0 0 0 0 0 0 0 4 E.faecalis Control 3 E. faecalis None 0 0 0 0 0 0 0 5 E. faecium Control 1E. faecium None 0 0 0 0 0 0 0 6 E. faecium Control 2 E. faecium None 0 00 0 0 0 0 7 E. faecium Control 3 E. faecium None 0 0 0 0 0 0 0 8 E.avium Control 1 E. avium None 0 0 0 0 0 0 0 9 E. avium Control 2 E.avium None 0 0 0 0 0 0 0 10 E. avium Control 3 E. avium None 0 0 0 0 0 00 11 E. casseliflavus Control 1 E. casseliflavus vanC 0 0 0 0 0 0 0 12E. casseliflavus Control 2 E. casseliflavus vanC 0 0 0 0 0 0 0 13 E.casseliflavus Control 3 E. casseliflavus vanC 0 0 0 0 0 0 0 14 E.casseliflavus Control 4 E. casseliflavus vanC 0 0 0 0 0 0 0 15 E.casseliflavus Control 5 E. casseliflavus vanC 0 0 0 0 0 0 0 16 E.gallinarum Control 1 E. gallinarum vanC 0 0 0 0 0 0 0 17 E. gallinarumControl 2 E. gallinarum vanC 0 0 0 0 0 0 0 18 E. gallinarum Control 3 E.gallinarum vanC 0 0 0 0 0 0 0 19 E. gallinarum Control 4 E. gallinarumvanC 0 0 0 0 0 0 0 20 E. gallinarum Control 5 E. gallinarum vanC 0 0 0 00 0 0 21 TW 3491 E. faecium vanA +++ +++ ++ + 55 5 0 22 TW 4558 E.faecium vanA +++ +++ ++ + 61 7 0 23 TW 4559 E. faecium vanA +++ +++ ++ +49 2 1 24 TW 4561 E. faecium vanA +++ +++ ++ + 35 2 0 25 TW 4590 E.faecium vanA +++ +++ ++ + 58 5 0 26 TW 3492 E. faecium vanB +++ +++ ++ +(44) 2 0 27 TW 4560 E. faecium vanB +++ +++ ++ + 22 5 0 28 TW 4589 E.faecalis vanB + 2 3 1 0 0 0 29 TW 5246 E. faecalis vanB +++ +++ ++ + 235 0 30 TW 5247 E. faecium vanB +++ ++ (+) 0 0 0 0 31 TW 5604 E. faeciumvanB +++ ++ (+) (+) 0 0 0 32 TW 5607 E. faecium vanB +++ +++ ++ + 28 1 033 TW 5608 E. faecalis vanB +++ +++ ++ + 12 1 0 34 TW 5609 E. faecalisvanB +++ +++ ++ + 33 2 0 35 TW 5610 E. faecalis vanB +++ ++ + 40  4 1 036 TW 5611 E. faecalis vanB (+) (+) (+) 0 0 0 0 37 TW 5645 E. faeciumvanB +++ +++ ++ + 36 5 0 38 TW 5646 E. faecium vanB +++ +++ ++ + 16 2 039 TW 5668 E. faecium vanB +++ + (+) 0 0 0 0 40 TW 5669 E. faecium vanB++ (+) (+) 0 0 0 0 41 TW 5670 E. faecium vanB +++ ++ (+) 0 0 0 0 42 TW5671 E. faecium vanB +++ ++ (+) 0 0 0 0 43 TW 5672 E. faecium vanB ++++++ ++ + 43 6 0 44 TW 5682 E. faecalis vanB +++ +++ ++ ++ + 28 5 45 TW5683 E. faecalis vanB +++ +++ ++ + 30 6 5 46 TW 5684 E. faecalis vanB+++ +++ ++ + 72 18 1 47 TW 5685 E. faecalis vanB +++ +++ ++ + 14 3 0 48TW 5686 E. faecalis vanB +++ +++ ++ + 68 19 1 49 TW 5687 E. faecalisvanB +++ +++ ++ + 23 7 0 50 TW 5688 E. faecium vanB +++ +++ ++ + 39 6 251 TW 5689 E. faecium vanB +++ +++ ++ + 42 6 0 52 TW 5690 E. faeciumvanB +++ +++ +++ ++ + 17 1 53 TW 6169 E. faecalis vanB +++ +++ ++ ++ +19 10 54 TW 6170 E. faecalis vanB 0 0 0 0 0 0 0 55 TW 6171 E. faeciumvanB ++ (+) (+) 1 0 0 0 56 TW 6172 E. faecium vanB (+) 0 0 0 0 0 0 57 TW6173 E. faecium vanB +++ +++ ++ + 47 11 4 58 TW 6174 E. faecium vanB ++++++ ++ + 40 3 2 59 TW 6175 E. faecium vanB +++ +++ ++ + 56 9 0 60 TW6176 E. faecium vanB +++ +++ ++ + 36 2 1 61 TW 6177 E. faecalis vanB ++++++ ++ + 49 13 1 62 TW 6178 E. faecium vanB +++ +++ ++ + 28 3 1 63 TW6179 E. faecalis vanB +++ ++ + 60  7 2 0 64 TW 6180 E. faecium vanB ++++++ ++ + 39 10 0 65 TW 6181 E. faecium vanB +++ +++ ++ + 20 3 1 66 TW6183 E. faecalis vanB +++ +++ ++ + 15 5 1 67 TW 6184 E. faecalis vanB+++ +++ ++ + 52 11 2 68 TW 6185 E. faecium vanB +++ +++ ++ (+) 0 5 0 69TW 7515 E. faecalis vanB +++ +++ ++ + 32 3 0 70 TW 7516 E. faecalis vanB+++ +++ ++ + 46 2 0

TABLE 5 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) BHI agar with 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, and 0.2 mol/l of sodium lactateDilution Resistance Undiluted Seq. Organism # Strains gene solution×10⁻¹ ×10⁻² ×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC 29212 E. faecalisNone 0 0 0 0 0 0 0 2 E. faecalis Control 1 E. faecalis None 0 0 0 0 0 00 3 E. faecalis Control 2 E. faecalis None 0 0 0 0 0 0 0 4 E. faecalisControl 3 E. faecalis None 0 0 0 0 0 0 0 5 E. faecium Control 1 E.faecium None 0 0 0 0 0 0 0 6 E. faecium Control 2 E. faecium None 0 0 00 0 0 0 7 E. faecium Control 3 E. faecium None 0 0 0 0 0 0 0 8 E. aviumControl 1 E. avium None 0 0 0 0 0 0 0 9 E. avium Control 2 E. avium None0 0 0 0 0 0 0 10 E. avium Control 3 E. avium None 0 0 0 0 0 0 0 11 E.casseliflavus Control 1 E. casseliflavus vanC 0 0 0 0 0 0 0 12 E.casseliflavus Control 2 E. casseliflavus vanC 0 0 0 0 0 0 0 13 E.casseliflavus Control 3 E. casseliflavus vanC 0 0 0 0 0 0 0 14 E.casseliflavus Control 4 E. casseliflavus vanC 0 0 0 0 0 0 0 15 E.casseliflavus Control 5 E. casseliflavus vanC 0 0 0 0 0 0 0 16 E.gallinarum Control 1 E. gallinarum vanC 0 0 0 0 0 0 0 17 E. gallinarumControl 2 E. gallinarum vanC 0 0 0 0 0 0 0 18 E. gallinarum Control 3 E.gallinarum vanC 0 0 0 0 0 0 0 19 E. gallinarum Control 4 E. gallinarumvanC 0 0 0 0 0 0 0 20 E. gallinarum Control 5 E. gallinarum vanC 0 0 0 00 0 0 21 TW 3491 E. faecium vanA +++ +++ ++ + 62 8 1 22 TW 4558 E.faecium vanA +++ +++ ++ + 66 9 1 23 TW 4559 E. faecium vanA +++ +++ ++ +60 5 0 24 TW 4561 E. faecium vanA +++ +++ ++ + 41 6 0 25 TW 4590 E.faecium vanA +++ +++ ++ + 68 5 0 26 TW 3492 E. faecium vanB +++ +++ ++ +46 10 2 27 TW 4560 E. faecium vanB +++ +++ ++ + 26 4 0 28 TW 4589 E.faecalis vanB +++ +++ ++ + 22 2 0 29 TW 5246 E. faecalis vanB +++ +++++ + 22 3 0 30 TW 5247 E. faecium vanB +++ +++ ++ + 36 3 0 31 TW 5604 E.faecium vanB +++ +++ ++ + 59 5 1 32 TW 5607 E. faecium vanB +++ +++ ++ +24 4 0 33 TW 5608 E. faecalis vanB +++ +++ ++ + 14 1 0 34 TW 5609 E.faecalis vanB +++ +++ ++ + 28 0 0 35 TW 5610 E. faecalis vanB +++ ++ +34  8 0 0 36 TW 5611 E. faecalis vanB +++ +++ ++ + 37 0 0 37 TW 5645 E.faecium vanB +++ +++ ++ + 43 3 1 38 TW 5646 E. faecium vanB +++ +++ ++ +23 2 1 39 TW 5668 E. faecium vanB +++ +++ ++ + 40 2 0 40 TW 5669 E.faecium vanB +++ +++ ++ + 46 2 0 41 TW 5670 E. faecium vanB +++ +++ ++ +44 7 0 42 TW 5671 E. faecium vanB +++ +++ ++ + 27 6 0 43 TW 5672 E.faecium vanB +++ +++ ++ + 32 9 3 44 TW 5682 E. faecalis vanB +++ +++ +++++ + 18 2 45 TW 5683 E. faecalis vanB +++ +++ ++ + 40 9 6 46 TW 5684 E.faecalis vanB +++ +++ ++ + 58 11 4 47 TW 5685 E. faecalis vanB +++ +++++ + 27 6 0 48 TW 5686 E. faecalis vanB +++ +++ ++ + 47 12 2 49 TW 5687E. faecalis vanB +++ +++ ++ + 30 4 1 50 TW 5688 E. faecium vanB +++ +++++ + 42 7 2 51 TW 5689 E. faecium vanB +++ +++ ++ + 26 3 0 52 TW 5690 E.faecium vanB +++ +++ ++ + 111 11 2 53 TW 6169 E. faecalis vanB +++ ++++++ ++ + 17 7 54 TW 6170 E. faecalis vanB +++ +++ ++ + 43 4 3 55 TW 6171E. faecium vanB +++ +++ ++ + 12 2 1 56 TW 6172 E. faecium vanB +++ +++++ + 17 6 1 57 TW 6173 E. faecium vanB +++ +++ ++ + 70 2 0 58 TW 6174 E.faecium vanB +++ +++ ++ + 57 13 2 59 TW 6175 E. faecium vanB +++ +++++ + 43 8 0 60 TW 6176 E. faecium vanB +++ +++ ++ + 73 9 1 61 TW 6177 E.faecalis vanB +++ +++ ++ + 43 6 4 62 TW 6178 E. faecium vanB +++ +++++ + 37 7 2 63 TW 6179 E. faecalis vanB +++ +++ ++ + 27 3 2 64 TW 6180E. faecium vanB +++ +++ ++ + 40 10 1 65 TW 6181 E. faecium vanB +++ +++++ + 31 2 0 66 TW 6183 E. faecalis vanB +++ +++ ++ + 17 2 1 67 TW 6184E. faecalis vanB +++ +++ ++ + 47 15 4 68 TW 6185 E. faecium vanB +++ +++++ + 68 13 8 69 TW 7515 E. faecalis vanB +++ +++ ++ ++ + 6 0 70 TW 7516E. faecalis vanB +++ +++ ++ + 46 3 1

TABLE 6 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) 5% sheep blood agar DilutionResistance Undiluted Seq. Organism # Strains gene solution ×10⁻¹ ×10⁻²×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC 29212 E. faecalis None ++++++ ++ + 30 4 0 2 E. faecalis Control 1 E. faecalis None +++ +++ ++ + 423 0 3 E. faecalis Control 2 E. faecalis None +++ +++ ++ + 51 3 0 4 E.faecalis Control 3 E. faecalis None +++ +++ ++ + 38 1 0 5 E. faeciumControl 1 E. faecium None +++ +++ ++ + 29 1 0 6 E. faecium Control 2 E.faecium None +++ +++ ++ + 40 5 0 7 E. faecium Control 3 E. faecium None+++ +++ ++ + 38 5 0 8 E. avium Control 1 E. avium None +++ +++ ++ + 21 10 9 E. avium Control 2 E. avium None +++ +++ ++ + 38 1 0 10 E. aviumControl 3 E. avium None +++ +++ ++ + 33 1 0 11 E. casseliflavus Control1 E. casseliflavus vanC +++ +++ ++ + 44 5 0 12 E. casseliflavus Control2 E. casseliflavus vanC +++ +++ ++ + 38 5 0 13 E. casseliflavus Control3 E. casseliflavus vanC +++ +++ ++ + 42 2 0 14 E. casseliflavus Control4 E. casseliflavus vanC +++ +++ ++ + 50 1 0 15 E. casseliflavus Control5 E. casseliflavus vanC +++ +++ ++ + 37 1 0 16 E. gallinarum Control 1E. gallinarum vanC +++ +++ ++ + 38 4 0 17 E. gallinarum Control 2 E.gallinarum vanC +++ +++ ++ + 61 7 0 18 E. gallinarum Control 3 E.gallinarum vanC +++ +++ ++ + 44 8 0 19 E. gallinarum Control 4 E.gallinarum vanC +++ +++ ++ + 51 4 0 20 E. gallinarum Control 5 E.gallinarum vanC +++ +++ ++ + 31 1 0 21 TW 3491 E. faecium vanA +++ +++++ + 60 10 0 22 TW 4558 E. faecium vanA +++ +++ ++ + 40 6 0 23 TW 4559E. faecium vanA +++ +++ ++ + 38 2 0 24 TW 4561 E. faecium vanA +++ +++++ + 51 5 0 25 TW 4590 E. faecium vanA +++ +++ ++ + 58 5 0 26 TW 3492 E.faecium vanB +++ +++ ++ + 100 13 1 27 TW 4560 E. faecium vanB +++ +++++ + 100 8 0 28 TW 4589 E. faecium vanB +++ ++ + 100 18 7 0 29 TW 5246E. faecium vanB +++ ++ + 100 20 7 1 30 TW 5247 E. faecium vanB +++ +++++ + 70 7 1 31 TW 5604 E. faecium vanB +++ +++ ++ + 60 10 1 32 TW 5607E. faecium vanB +++ +++ ++ + 100 19 3 33 TW 5608 E. faecalis vanB +++++ +  80 20 20 1 34 TW 5609 E. faecalis vanB +++ ++ +  70 8 6 1 35 TW5610 E. faecalis vanB +++ ++ + 100 50 50 4 36 TW 5611 E. faecalis vanB+++ ++ + 100 50 50 8 37 TW 5645 E. faecium vanB +++ +++ ++ + 30 10 3 38TW 5646 E. faecium vanB +++ +++ ++ + 40 25 4 39 TW 5668 E. faecium vanB+++ +++ ++ + 50 5 1 40 TW 5669 E. faecium vanB +++ +++ ++ + 100 9 1 41TW 5670 E. faecium vanB +++ +++ ++ + 70 9 1 42 TW 5671 E. faecium vanB+++ +++ ++ + 70 10 3 43 TW 5672 E. faecium vanB +++ +++ ++ + 17 1 0 44TW 5682 E. faecalis vanB +++ +++ ++ + 80 8 0 45 TW 5683 E. faecalis vanB+++ +++ ++ + 60 1 1 46 TW 5684 E. faecalis vanB +++ +++ ++ + 13 6 0 47TW 5685 E. faecalis vanB +++ ++ +  40 7 1 0 48 TW 5686 E. faecalis vanB+++ ++ +  60 8 2 1 49 TW 5687 E. faecalis vanB +++ + 100  8 2 0 0 50 TW5688 E. faecium vanB +++ +++ ++ + 30 5 0 51 TW 5689 E. faecium vanB ++++++ ++ + 30 4 2 52 TW 5690 E. faecium vanB +++ +++ ++ + 50 10 0 53 TW6169 E. faecalis vanB +++ ++ +  40 19 22 8 54 TW 6170 E. faecalis vanB+++ +++ ++ + 24 2 1 55 TW 6171 E. faecium vanB +++ +++ ++ + 60 8 1 56 TW6172 E. faecium vanB +++ +++ ++ + 40 3 0 57 TW 6173 E. faecium vanB ++++++ ++ + 40 5 3 58 TW 6174 E. faecium vanB +++ +++ ++ + 60 11 4 59 TW6175 E. faecium vanB +++ +++ ++ + 25 1 2 60 TW 6176 E. faecium vanB ++++++ ++ + 50 6 2 61 TW 6177 E. faecalis vanB +++ +++ ++ + 20 18 3 62 TW6178 E. faecium vanB +++ +++ ++ + 40 4 0 63 TW 6179 E. faecalis vanB+++ + 100  10 1 2 0 64 TW 6180 E. faecium vanB +++ +++ ++ + 40 9 1 65 TW6181 E. faecium vanB +++ +++ ++ + 50 8 5 66 TW 6183 E. faecalis vanB +++++ +  40 14 4 0 67 TW 6184 E. faecalis vanB +++ ++ + 100 13 3 0 68 TW6185 E. faecium vanB +++ +++ ++ + 60 2 0 69 TW 7515 E. faecalis vanB +++++ + 100 30 4 1 70 TW 7516 E. faecalis vanB +++ ++ +  80 15 5 0

TABLE 7 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) Enterococcosel agar with 8 μg/mlof vancomycin Dilution Resistance Undiluted Seq. Organism # Strains genesolution ×10⁻¹ ×10⁻² ×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC 29212 E.faecalis None 0 0  0  0 0 0 0 2 E. faecalis Control 1 E. faecalis None 00  0  0 0 0 0 3 E. faecalis Control 2 E. faecalis None 0 0  0  0 0 0 0 4E. faecalis Control 3 E. faecalis None 0 0  0  0 0 0 0 5 E. faeciumControl 1 E. faecium None 0 0  0  0 0 0 0 6 E. faecium Control 2 E.faecium None 0 0  0  0 0 0 0 7 E. faecium Control 3 E. faecium None 0 0 0  0 0 0 0 8 E. avium Control 1 E. avium None 0 0  0  0 0 0 0 9 E.avium Control 2 E. avium None 0 0  0  0 0 0 0 10 E. avium Control 3 E.avium None 0 0  0  0 0 0 0 11 E. casseliflavus Control 1 E.casseliflavus vanC +++ +++ ++ + (41) 5 0 12 E. casseliflavus Control 2E. casseliflavus vanC +++ +++ ++ + 32 3 0 13 E. casseliflavus Control 3E. casseliflavus vanC +++ +++ ++ + 21 3 0 14 E. casseliflavus Control 4E. casseliflavus vanC +++ +++ ++ + 26 3 0 15 E. casseliflavus Control 5E. casseliflavus vanC +++ +++ ++ + 33 1 0 16 E. gallinarum Control 1 E.gallinarum vanC +++ +++ ++ + 33 4 0 17 E. gallinarum Control 2 E.gallinarum vanC +++ +++ ++ + 48 4 0 18 E. gallinarum Control 3 E.gallinarum vanC +++ +++ ++ + 29 6 0 19 E. gallinarum Control 4 E.gallinarum vanC +++ +++ ++ + 40 2 0 20 E. gallinarum Control 5 E.gallinarum vanC +++ +++ ++ + 35 1 0 21 TW 3491 E. faecium vanA +++ +++++ + 55 7 0 22 TW 4558 E. faecium vanA +++ +++ ++ + 47 6 0 23 TW 4559 E.faecium vanA +++ +++ ++ + 52 6 0 24 TW 4561 E. faecium vanA +++ +++ ++ +48 2 0 25 TW 4590 E. faecium vanA +++ +++ ++ + 51 2 0 26 TW 3492 E.faecium vanB +++ +++ ++ + 100 12 0 27 TW 4560 E. faecium vanB + (+) 1  00 0 0 28 TW 4589 E. faecalis vanB +++ ++ + 100  18 2 2 29 TW 5246 E.faecalis vanB +++ ++ + 100  40 4 1 30 TW 5247 E. faecium vanB +++ +++++ + 70 7 0 31 TW 5604 E. faecium vanB +++ +++ (+) (200) 0 0 0 32 TW5607 E. faecium vanB +++ +++ ++ + 100 20 5 33 TW 5608 E. faecalis vanB+++ ++ + 80 19 12 1 34 TW 5609 E. faecalis vanB +++ ++ + 70 6 15 0 35 TW5610 E. faecalis vanB +++ ++ + 100  50 40 5 36 TW 5611 E. faecalis vanB+++ ++ + 100  30 20 2 37 TW 5645 E. faecium vanB +++ +++ ++ + 30 8 1 38TW 5646 E. faecium vanB +++ +++ ++ + 40 25 1 39 TW 5668 E. faecium vanB+++ +++ ++ + 50 7 0 40 TW 5669 E. faecium vanB +++ +++ ++ + 100 4 1 41TW 5670 E. faecium vanB +++ +++ ++ + 60 10 1 42 TW 5671 E. faecium vanB+++ ++ + 12 1 0 0 43 TW 5672 E. faecium vanB +++ +++ ++ + 17 2 0 44 TW5682 E. faecalis vanB +++ +++ ++ + 40 1 1 45 TW 5683 E. faecalis vanB+++ +++ ++ + 30 3 2 46 TW 5684 E. faecalis vanB +++ +++ ++ + 25 6 0 47TW 5685 E. faecalis vanB +++ ++ + 40 2 0 0 48 TW 5686 E. faecalis vanB+++ ++ + 20 1 0 0 49 TW 5687 E. faecalis vanB ++ + 100  4 0 0 0 50 TW5688 E. faecium vanB +++ ++ + (+) (30) 0 0 51 TW 5689 E. faecium vanB+++ +++ ++ + 30 1 1 52 TW 5690 E. faecium vanB +++ +++ ++ + 50 6 1 53 TW6169 E. faecalis vanB +++ ++ + 30 15 13 1 54 TW 6170 E. faecalis vanB+++ ++ + 60 14 1 0 55 TW 6171 E. faecium vanB +++ +++ ++ + 30 17 2 56 TW6172 E. faecium vanB +++ ++ + (100) (20) (5) 0 57 TW 6173 E. faeciumvanB +++ +++ ++ + 46 6 2 58 TW 6174 E. faecium vanB +++ (+) (40) 0 0 0 059 TW 6175 E. faecium vanB +++ +++ ++ + 21 6 0 60 TW 6176 E. faeciumvanB +++ +++ ++ + 50 7 0 61 TW 6177 E. faecalis vanB +++ +++ ++ + 30 206 62 TW 6178 E. faecium vanB +++ +++ ++ + 30 4 0 63 TW 6179 E. faecalisvanB ++ + 50 5 1 0 0 64 TW 6180 E. faecium vanB +++ (++) (+) 1 0 0 0 65TW 6181 E. faecium vanB +++ +++ ++ + 50 11 2 66 TW 6183 E. faecalis vanB+++ ++ + 40 4 1 0 67 TW 6184 E. faecalis vanB +++ ++ + 100  18 1 0 68 TW6185 E. faecium vanB +++ +++ ++ + 40 4 2 69 TW 7515 E. faecalis vanB +++++ + 100  20 1 0 70 TW 7516 E. faecalis vanB ++ + 40 4 1 0 0

TABLE 8 Number of colony growth on each medium in a dilution culturetest (incubated at 35° C. for 48 hours) BHI agar with 32 μg/ml ofvancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l of DL-sodium lactate,20 μg/ml of polymyxin B, 20 μg/ml of aztreonam, 2 μg/ml of amphotericinB, 50 μg/ml of TTC Dilution Resistance Undiluted Seq. Organism # Strainsgene solution ×10⁻¹ ×10⁻² ×10⁻³ ×10⁻⁴ ×10⁻⁵ ×10⁻⁶ 1 E. faecalis ATCC29212 E. faecalis None 0 0  0  0 0 0 0 2 E. faecalis Control 1 E.faecalis None 0 0  0  0 0 0 0 3 E. faecalis Control 2 E. faecalis None 00  0  0 0 0 0 4 E. faecalis Control 3 E. faecalis None 0 0  0  0 0 0 0 5E. faecium Control 1 E. faecium None 0 0  0  0 0 0 0 6 E. faeciumControl 2 E. faecium None 0 0  0  0 0 0 0 7 E. faecium Control 3 E.faecium None 0 0  0  0 0 0 0 8 E. avium Control 1 E. avium None 0 0  0 0 0 0 0 9 E. avium Control 2 E. avium None 0 0  0  0 0 0 0 10 E. aviumControl 3 E. avium None 0 0  0  0 0 0 0 11 E. casseliflavus Control 1 E.casseliflavus vanC 0 0  0  0 0 0 0 12 E. casseliflavus Control 2 E.casseliflavus vanC 0 0  0  0 0 0 0 13 E. casseliflavus Control 3 E.casseliflavus vanC 0 0  0  0 0 0 0 14 E. casseliflavus Control 4 E.casseliflavus vanC 0 0  0  0 0 0 0 15 E. casseliflavus Control 5 E.casseliflavus vanC 0 0  0  0 0 0 0 16 E. gallinarum Control 1 E.gallinarum vanC 0 0  0  0 0 0 0 17 E. gallinarum Control 2 E. gallinarumvanC 0 0  0  0 0 0 0 18 E. gallinarum Control 3 E. gallinarum vanC 0 0 0  0 0 0 0 19 E. gallinarum Control 4 E. gallinarum vanC 0 0  0  0 0 00 20 E. gallinarum Control 5 E. gallinarum vanC 0 0  0  0 0 0 0 21 TW3491 E. faecium vanA +++ +++ ++ + 62 11 0 22 TW 4558 E. faecium vanA ++++++ ++ + 43 2 0 23 TW 4559 E. faecium vanA +++ +++ ++ + 42 2 0 24 TW4561 E. faecium vanA +++ +++ ++ + 60 3 0 25 TW 4590 E. faecium vanA ++++++ ++ + 59 6 0 26 TW 3492 E. faecium vanB +++ +++ ++ + 100 13 2 27 TW4560 E. faecium vanB +++ +++ ++ + 50 4 2 28 TW 4589 E. faecalis vanB +++++ + 100  30 10 0 29 TW 5246 E. faecalis vanB +++ ++ + 100  30 2 0 30 TW5247 E. faecium vanB +++ +++ ++ + 60 7 1 31 TW 5604 E. faecium vanB ++++++ ++ + 80 8 3 32 TW 5607 E. faecium vanB +++ +++ ++ + 100 12 1 33 TW5608 E. faecalis vanB +++ ++ + 60 25 13 2 34 TW 5609 E. faecalis vanB+++ ++ + 70 10 1 0 35 TW 5610 E. faecalis vanB +++ ++ + 100  50 50 8 36TW 5611 E. faecalis vanB +++ ++ + 100  70 60 10 37 TW 5645 E. faeciumvanB +++ +++ ++ + 30 3 1 38 TW 5646 E. faecium vanB +++ +++ ++ + 40 25 339 TW 5668 E. faecium vanB +++ +++ ++ + 50 5 1 40 TW 5669 E. faeciumvanB +++ +++ ++ + 100 6 2 41 TW 5670 E. faecium vanB +++ +++ ++ + 80 2 142 TW 5671 E. faecium vanB +++ +++ ++ + 50 5 0 43 TW 5672 E. faeciumvanB +++ +++ ++ + 30 0 0 44 TW 5682 E. faecalis vanB +++ +++ ++ + 60 6 045 TW 5683 E. faecalis vanB +++ +++ ++ + 40 1 0 46 TW 5684 E. faecalisvanB +++ +++ ++ + 20 7 0 47 TW 5685 E. faecalis vanB +++ ++ + 40 1 0 048 TW 5686 E. faecalis vanB +++ ++ + 40 3 0 1 49 TW 5687 E. faecalisvanB ++ + 50 7 0 0 0 50 TW 5688 E. faecium vanB +++ +++ ++ + 40 3 4 51TW 5689 E. faecium vanB +++ +++ ++ + 40 4 0 52 TW 5690 E. faecium vanB+++ +++ ++ + 40 9 0 53 TW 6169 E. faecalis vanB +++ ++ + 40 28 15 4 54TW 6170 E. faecalis vanB +++ ++ + 100  30 2 0 55 TW 6171 E. faecium vanB+++ ++ + 100  30 5 0 56 TW 6172 E. faecium vanB +++ +++ ++ + 30 7 2 57TW 6173 E. faecium vanB +++ +++ ++ + 40 7 2 58 TW 6174 E. faecium vanB+++ +++ ++ + 50 5 2 59 TW 6175 E. faecium vanB +++ +++ ++ + 20 1 1 60 TW6176 E. faecium vanB +++ +++ ++ + 50 15 2 61 TW 6177 E. faecalis vanB+++ +++ ++ + 20 11 2 62 TW 6178 E. faecium vanB +++ +++ ++ + 30 7 0 63TW 6179 E. faecalis vanB ++ + 50  6 1 0 0 64 TW 6180 E. faecium vanB ++++++ ++ + 40 8 3 65 TW 6181 E. faecium vanB +++ +++ ++ + 50 8 5 66 TW6183 E. faecalis vanB +++ ++ + 40 9 1 1 67 TW 6184 E. faecalis vanB +++++ + 100  13 1 1 68 TW 6185 E. faecium vanB +++ +++ ++ + 40 4 0 69 TW7515 E. faecalis vanB +++ ++ + 100  30 2 0 70 TW 7516 E. faecalis vanB++ + 100  15 2 0 0

TABLE 9 Strains detected in VRE screening of actual fecal specimensusing screening media (incubated at 35° C. for 48 hours) BHI agar with32 μg/ml of vancomycin, 32 μg/ml of D-cycloserine, 0.2 mol/l DL-sodiumlactete, Enterococcosel agar with 20 μg/ml of polymyxin B, 20 μg/ml ofaztreonam, 8 μg/ml of vancomycin 2 μg/ml of amphotericin B and 50 μg/mlTTC Number of specimens 13 pecimens (26.5%) 0 specimen (0%) where van CVRE is detected Number of specimens  0 specimen (0%)   0 specimen (0%)where van A and van B VRE are detected

TABLE 10 Number of Van B VRE detected in VRE screening of Van B VREinoculated fecal specimens using screening media (incubated at 35° C.for 48 hours) Fecal specimens BHI agar with 32 μg/ml of vancomycin, 32μg/ml to which Van B VRE of D-cycloserine, 0.2 mol/l DL-sodium lactete,Van B VRE used for Amount of Enterococcosel agar with 20 μg/ml ofpolymyxin B, 20 μg/ml of aztreonam, is inoculated inoculation inoculum 8μg/ml of vancomycin 2 μg/ml of amphotericin B and 50 μg/ml TTC Sample 1TW 4560 10~20 cells/10 μl Can not differentiate 16 cells/10 μl due tonumerous Van C VRE TW 6174 10~20 cells/10 μl Can not differentiate 13cells/10 μl due to numerous Van C VRE TW 6180 10~20 cells/10 μl Can notdifferentiate 17 cells/10 μl due to numerous Van C VRE Sample 2 TW 456010~20 cells/10 μl 15 cells/10 μl 10 cells/10 μl TW 6174 10~20 cells/10μl  0 cells/10 μl 18 cells/10 μl TW 6180 10~20 cells/10 μl 15 cells/10μl 15 cells/10 μl

1. A Van A and Van B vancomycin resistant enterococci detection mediumwhere vancomycin, D-cycloserine and D-lactate are added to a culturemedium formula where enterococci can grow.
 2. A Van A and Van Bvancomycin resistant enterococci detection medium where 32-256 μg/ml ofvancomycin, 1-64 μg/ml of D-cycloserine and 0.025-0.8 mol/l of DL-sodiumlactate are added to a culture medium formula where enterococci cangrow.
 3. A method of selectively detecting Van A and Van B vancomycinresistant enterococci, comprising inoculating a suspension of a testingmicroorganism to a Van A and Van B vancomycin resistant enterococcidetection medium of claim 1, and incubating the medium at 35-37° C. for24-48 hours.
 4. A method of selectively detecting Van A and Van Bvancomycin resistant enterococci, comprising inoculating a specimen to aVan A and Van B vancomycin resistant enterococci detection medium ofclaim 1, and incubating the medium at 35-37° C. for 24-48 hours.
 5. Amethod of selectively detecting Van A and Van B vancomycin resistantenterococci, comprising inoculating suspension of a testingmicroorganism to a Van A and Van B vancomycin resistant enterococcidetection medium of claim 2, and incubating the medium at 35-37° C. for24-48 hours.
 6. A method of selectively detecting Van A and Van Bvancomycin resistant enterococci, comprising inoculating a specimen to aVan A and Van B vancomycin resistant enterococci detection medium ofclaim 2, and incubating the medium at 35-37° C. for 24-48 hours.
 7. Amethod of selectively detecting Van A and Van B vancomycin resistantenterococci, comprising inoculating a suspension of a testingmicroorganism to a Van A and Van B vancomycin resistant detection mediumaccording to claim 1, incubating said medium, and determining whetherVan A and Van B vancomycin resistant enterococci are present in saiddetection medium.
 8. A method of selectively detecting Van A and Van Bvancomycin resistant enterococci, comprising inoculating a suspension ofa testing microorganism to a Van A and Van B vancomycin resistantdetection medium according to claim 2, incubating said medium, anddetermining whether Van A and Van B vancomycin resistant enterococci arepresent in said detection medium.